Identification and characteristics of the structural gene for the Drosophila eye colour mutant sepia, encoding PDA synthase, a member of the omega class glutathione S-transferases.

School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.

Abstract

The eye colour mutant sepia (se1) is defective in PDA {6-acetyl-2-amino-3,7,8,9-tetrahydro-4H-pyrimido[4,5-b]-[1,4]diazepin-4-one or pyrimidodiazepine} synthase involved in the conversion of 6-PTP (2-amino-4-oxo-6-pyruvoyl-5,6,7,8-tetrahydropteridine; also known as 6-pyruvoyltetrahydropterin) into PDA, a key intermediate in drosopterin biosynthesis. However, the identity of the gene encoding this enzyme, as well as its molecular properties, have not yet been established. Here, we identify and characterize the gene encoding PDA synthase and show that it is the structural gene for sepia. Based on previously reported information [Wiederrecht, Paton and Brown (1984) J. Biol. Chem. 259, 2195-2200; Wiederrecht and Brown (1984) J. Biol. Chem. 259, 14121-14127; Andres (1945) Drosoph. Inf. Serv. 19, 45; Ingham, Pinchin, Howard and Ish-Horowicz (1985) Genetics 111, 463-486; Howard, Ingham and Rushlow (1988) Genes Dev. 2, 1037-1046], we isolated five candidate genes predicted to encode GSTs (glutathione S-transferases) from the presumed sepia locus (region 66D5 on chromosome 3L). All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro. The molecular mass of recombinant CG6781 was estimated to be 28 kDa by SDS/PAGE and 56 kDa by gel filtration, indicating that it is a homodimer under native conditions. Sequencing of the genomic region spanning CG6781 revealed that the se1 allele has a frameshift mutation from 'AAGAA' to 'GTG' at nt 190-194, and that this generates a premature stop codon. Expression of the CG6781 open reading frame in an se1 background rescued the eye colour defect as well as PDA synthase activity and drosopterins content. The extent of rescue was dependent on the dosage of transgenic CG6781. In conclusion, we have discovered a new catalytic activity for an Omega class GST and that CG6781 is the structural gene for sepia which encodes PDA synthase.

Genetic and cytological map of chromosome 3L showing sepia and its environs

The fact that sepia is included in the deletion in Tp(3;3)hM3 predicts that the sepia gene is located in the interval 66D1 to 66D10 on the cytological map. The upper and lower solid lines indicate the genetic and cytological locations of the five candidate CGs of PDA synthase found in the 66D5 region. The open box represents the deletion that includes the sepia gene, and the filled box the material transposed in Tp(3;3)hM3. The dotted vertical lines indicate the limits of the chromosomal deletion, while the solid oblique lines indicate their limits on the cytogenetic map. jv (javelin), se (sepia) and h (hairy) have been mapped by recombination to 3–19.2, 3–26.0 and 3–26.5 respectively. The breakpoints of Tp(3;3)hM3 include 61E, 66D1−3 and 66D10 on the cytological map, and its 66D10 break is located at −10.8 to −9.35 kb on the DNA map of h.

The five candidates for PDA synthase were predicted to be Omega class GSTs using the InterPro program. Multiple sequence alignment of the candidates at 66D5 on the cytological map was performed with human GSTO1 (hGSTO1), human GSTO2 (hGSTO2), mouse GSTO1 (mGSTO1) and rat GSTO1 (rGSTO1) using the ClustalW program. Identical and semi-conserved amino acids are highlighted in black and grey respectively. The arrowhead indicates the putative binding site for the substrate GSH which is characteristic of Omega class GSTs. A % identity/% similarity matrix is shown at the bottom.

(A) Nucleotide and deduced amino acid sequence of CG6781. The sequence of PDA synthase (CG6781) is based on the Drosophila Genome Annotation (release 4.1). CG6781 is composed of three exons, two introns and short untranslated regions at each end. The deduced amino acid sequence is shown above the nucleotide sequence. Lower-case letters represent the untranslated region of the gene. Start and stop codons are shown in boldface, and a putative polyadenylation signal is underlined and in italics. The right-hand side of the sequence gives both nucleotide and amino acid numbering. The beginning of each exon is indicated by downward-pointing arrows. The pair of primers used for genomic sequencing is underlined. (B) The mutation in the coding region of CG6781 in the se1 allele. The cDNA sequence of the coding region of the second exon from 190 to 276 (numbered from the ATG translation initiation codon) is shown. Single-letter amino acid abbreviations are shown above the nucleotide sequence. In se1, nt 190–194, AAGAA, are changed to GTG, which leads to a frameshift mutation. This frameshift mutation produces a stop codon (TGA, underlined) at nt 273–275 in the second exon.

The coding region of CG6781 was cloned into pUAST, which was then used to generate UAS-CG6781 transgenic flies by microinjection. (A) Eye colour phenotypes of wild-type (Oregon-R), sepia mutant (y;;se1), flies with one copy each of GMR-gal4 and UAS-CG6781 in the se1 background (y;GMR-gal4 UAS-CG6781/+;se1), and flies with one copy of GMR-gal4 and two copies of UAS-CG6781 in the se1 background (y;GMR-gal4 UAS-CG6781/UAS-CG6781;se1). (B) PDA content (grey bars) and PDA synthase activity (white bars) of adult fly heads of each genotype were determined by HPLC and enzyme assays respectively. (C) The drosopterin content of adult fly heads of each genotype was determined spectrophotometrically as described in the Experimental section. The extent of rescue defined by the content of PDA and PDA synthase activity (B) or of drosopterin (C) is dependent on the dosage of UAS-CG6781. Levels are calculated relative to wild-type flies. The vertical bars represent means±S.E.M.